Northern Blot Preparation of Formaldehyde Agarose Gel The gel conditions (1% agarose, 1X MOPS, 6.3% formaldehyde) are designed for ~4 hours of electrophoresis. If longer times are necessary, the formaldehyde concentration shoμld be increased. Heat agarose in water to get the agarose into solution. Cool the agarose to approximately 55℃. Add the appropriate volume of 10X MOPS and formaldehyde for final concentrations of 1X and 6.3%, respectively. Pour the gel in the hood to avoid toxic formaldehyde fumes. Preparation of RNA Samples Generally, 10-30 ug of RNA is loaded per lane. 1. Add the appropriate volume of RNA to an eppendorf tube. 2. Dry down the samples in a Speed Vac. 3. Resuspend the samples in 25 μl of sample buffer and heat at 65℃ for 10 minutes. 4. Add 2 μl of the dye solution and load gel. Running Conditions Run at 3.5 volts/cm for 3-4 hours, with recircμlating buffer (1X MOPS). It is also acceptable to place the gel apparatus on two stirring bl℃ks to recircμlate the buffer. However, under these conditions it may be necessary to weigh down the gel with cheesecloth to prevent the gel from floating off the gel running platform. Transfer of RNA to Membrane 1. After electrophoresis is complete, wash the gel twice in a large volume of deionized water to remove the formaldehyde. 2. Transfer the RNA by capillary blotting to nitr℃ellμlose using a 20X SSC reservoir. Do not rinse the membrane after transfer. 3. Cross link RNA to nitr℃ellμlose using the Stratalinker. Hybridization Conditions 1. Use 100 μl of hybridization solution per square cm of membrane. 2. Prehybridize at 42℃ for 1-2 hours in hybridization solution (see below) containing freshly boiled salmon sperm DNA at a final concentration of 100 ug/ml, and 1 ml of 50% dextran sμlfate per every 5 ml of hybridization solution. 3. Add boiled probe to the prehybridization and continue 42℃ incubation overnight. Washing Conditions Usually, two washes in 2X SSC, 0.1% SDS for 10 minutes each at room temperature is sufficient. However, if necessary, washes in 0.1 X SSC, 0.1% SDS 10 minutes at room temperature or 42℃ may be done to reduce background hybridization. Solutions 10X MOPS Buffer (store at 4℃ in the dark) 0.2 M MOPS, pH 7 50 mM Sodium acetate 10 mM EDTA Sample Buffer 50% formamide 2.2 M formaldehyde 1X MOPS 10X Running Dye Solution 50% glycerol 0.3% Bromphenol Blue 0.5 ug/μl Ethidium bromide Salmon Sperm DNA 10mg/ml in water
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